Instrumentation Techniques (5-5) Calibration and Validation 953 Validation ation of Analytical Procedures: Text And a : : ‘al Procedures: Text And Methodology @ (RI). In part I of this guideline, text on Validation of Anal ical procedures has been discussed wherein, introduction, types of analyti 8 | 1n 2005, ICH published the guidetines on Vatid i idati cal procedures to be ted and valida ical procedures to be validat tion parameters / characteristics to be performed have been described in Part II. . ana < ee Procedures : Methodology, each validation parameter was — in detail. Method of conducting each parameter, number of samples to be taken, statistical parameters to be performed and their recommendations have been discussed. USFDA Published uate for Industry Q2B Validation of Analytical Procedures: Methodology which is complementary to ICH guidelines Q2 (RL). - Validation is a process of establishing documentary evidence demonstrating that a procedure, process, or activity carried out in production or testing maintains the desired : level of compliance at all stages. OR Validation is establishing documented evidence which provides a high degree of assurance duce a product meeting its pre-determined ° that a specific process will consistently pro specifications and quality characteristics. sed to confirm that the analytical procedure employed for Method validation is the process u: from method validation can be used to suitable for its intended use. Results reliability and consistency of analytical resi ° a specific test is s judge the quality, any good analytical practice. The objective of validation of an analytical proc uults; it is an integral part of edure is to demonstrate that it is suitable for . its intended purpose. »>| 5.3.1 Analytical Method Validation Types of analytical procedures to be validated are : 1, Identification tests 2, Quantitative tests for impurities content 3. Limit tests for the control of impurities 4. Quantitative tests of the active moiety in samples of drug substance or drug product TECHNICAL PUBLICATIONS® - an yp-thrust for knowledgeAdvanced Instrumentation Techniques (5-6) Calibration and Validation ¢ Analytical method validation parameters/characteristics to be performed as per ICH and USP are as follows - ICH USP A. | Specificity Specificity B. | Linearity Linearity C. | Range Range D. | Accuracy Accuracy E. | Precision Precision F. | Limit of detection Limit of detection G. | Limit of quantitation Limit of quantitation H. | Robustness Ruggedness I | System Suitability Robustness @ The detailed description of analytical method validation characteristics / parameters is as follows - A. Specificity © Itis the ability to assess unequivocally the analyte in the presence of components whichA. Specificity * Itis the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix, etc.). ¢ It is not always possible to demonstrate that an analytical procedure is specific for a Particular analyte. In this case a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination. a. Identification : le Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the analyte. 2. Assay—and—Impurity Test(s) : For chromatographic Procedures, representative chromatograms should be used to demonstrate specificity. The approach is similar for both assay and impurity tests. x, TECHNICAL PUBLICATIONS® - an up-thrust for knowledgeChapter 4... Parameters of Inst Contents ... cf 4.1” Techniques, Methods, Procedures an 4.2 Selecting an Analytical Method 4.2.1 Accuracy 4.2.2. Precision 4.3.3. Sensitivity 4.2.4. Selectivity 4.2.5 Robustness and Ruggedness 4.2.6 Scale of Operation 4.2.7 Equipment, Time and Cost 4.2.8 Making the Final Choice 4.3 Developing the Procedure 4.3.1 Calibration-and Standardization 4.3.2 Sampling 4.3.3. Validation 4.4 Protocols * Summary rumental Analysj, ‘otocols = Exercise Introduction ___ The analysis Provides the chemical and physical information about a sample. The analyte isa component of interest in the sample, while the matrix is the remainder of the sample. In any analysis, we determine the identity, properties and quantity of each constituent. : *hniques, Methods, Procedures and Protocols n any analysis, first we have to underst: i a approach this problem. To solve the: otal oe es Problem or analysis through f (3) Set the procedure and apply i accept results,‘Analytical Chemistry - I (Sem. v) 42 Parameters of Instrumental Analysis Example: Determination of Pb from water by AAS (Atomic Absorption Spectroscopy) 1 Techniques =[——*| Graphite furnace AAS 2 Methods d q L Pb in water | Pb in soil Pb in blood 3. [Procedures K-—| Ee feel ‘American Public Health Association —f American society of testing material Environmental protection agency 4, Protocols Fig. 4.1: Steps for problem solving - Determination of Pb by AAS A chemical or physical principle that can be used to analyze a sample is called as a technique. For the determination of a specific analyte, a specific: method is selected. In the above example, AAS technique is used and another method is used or selected for the determination of Pb from water, soil and blood respectively. The analyst should select best method for the particular analyte. A procedure is a set of written directions in detail on how to apply a method to a parti- cular analyte with all information about sampling, handling, solution preparation, interfere- nce to validation of results. There are many procedures available for the analysis of a parti- cular sample. The protocol is a set of stringent written guidelines detailing the procedure and it must be followed by the agency giving the protocol. This Protocol is to accept the result of the experiment. In the above example, the Pb level in water under the Safe Drinking Water Act labs follow a protocol specified by Environmental Protection Agency. 4.2 Selecting an Analytical Method Analytical Chemistry deals with analysis by separation, identification and determination of ® specific compound present in the sample. It splits into qualitative and quantitative analysis Methods, et ree analysis gives information about the presence of the constitution in the preach - fe quantitative analysis gives information about the quantity of each constituent WUaltative = sample. Far analysis, various methods are available. The qualitative Manjari * Organic qualitative, and general qualitative) methods are used for analysis, while Various ‘itati ‘i i erm ttative (Gravimetric, volumetric, instrumental, spectral etc.) methods are used for nation of analyte from the sample.of I [Link]. Analytical Chemistry - 1 (Sem. V) 43 Parameters of Instrumental ana Examples: ie 1. Gravimetric method for determination of Pb as precipitate of on OF as Pacy ! determination of Ni as Ni-DMG precipitate, Ba as BaSO, precipitate, Cl as Age} precipitate, Fe as a Fe;0; etc. ; : 2. Volumetric analysis for determination of the concentration acid or base neutralization titration, Zn as a Zn-EDTA complex (i.e. complexometric titration) redox titration etc. . 3. Spectral methods like colorimetric determination, spectrophotometric determination (R, UV, NMR) etc. 4. Conductometric, potentiometric, pH metric, and voltammetric methods etc, are available. ‘ jee The analyst has to choose a best method for the qualitative and quantitative method fo, ‘the determination of a specific method. While selecting analytical method, consideration is given to some or all the criteria like accuracy, precision, sensitivity, selectivity, robustness ang ruggedness, scale of operation, analysis time, availability of equipment and cost of analysis, 4.2.1 Accuracy The accuracy is the degree of agreement between measured value and the true value of the measurement i.e. consistency or concordance between the measured value and the true value or most probable value. It is measure of how closely the result of an experiment agrees with the expected result. The accuracy is mostly expressed in terms of relative error or percent relative error. Absolute error = Obtained result - Expected result Absolute error Expected/True value % Error = Relative error x 100 Analytical method is classified as; 1. Relative error is < 1% then analytical method is highly accurate 2. Relative error is in between 1% and 5% then analytical method is moderately accurate. Relative error = 3. Relative error is > 5% then method is of low accuracy. 4.2.2 Precision Precision is the degree of agreement between the reproducibility and repeatability of measurement or result. When the sample is analyzed several times, individual results are rarely the same. Precision is the measure of the variabil agreement between individual analyses, the more will precise the results. The precision is measured by calculating mean deviation, relative mean deviation, standard deviation, and Coefficient of variation. The precision level can be understood with the help of Fig. 4.2. The results of Mg in ppm are given in two ways. eeuAW Parameters of instrumental A pysse Amba Chemistry - 1 (Sem. v) 44 : yBSe alysis | “Sr 82s a ae ah as More precise ™g (ppm) ——» (a) x TT q 3 32 33 34 35 ‘ees procise mg (ppm) —e it) Fig. 4.2: Concentration of Mg in ) Ppm showing the effect of precision The precision is measured in terms of: s Xj 1, Arithmetic mean: Arithmetical mean X = Ex nis the number of observations, X; is the observation, 2. Deviation (A): It is the numerical differenc e between an individual result and the mean or average value of the result. A = |X-X| ol Mean or Average deviation (A): is the average deviation of each result. 5 Ay + Ap + Ag + Ag + ou. + Ay 4. Relative mean deviation: It is th average value of result in %. RMD = |41. 100 x 5. Standard deviation (s); It is the square root of the sum of the squares of the deviation divided by one less than the number of observations. A+ Ae Ast Hae Di Ki -X? ut n-1 7 n-1 n !@ ratio of mean or average deviation to the mean or 6. Coefficient of variation (C.V.): The standard deviation is expressed in relative standard deviation (S,) in %. 10 5, = cv, = 51000 x 4.3.3 Sensitivity Th many analyses, we use two samples with different amounts of analyte. The selected method can measure i its ability to detect the difference, Therefore, the sensitivity is a measure of the method Hl S ability to distinguish between two samples, reportable as the change in observation per unit change in the amount of analyte.te if [Link]. Analytical Chemistry - I (Sem. V) 45 Parameters of Instrumenta, Anal k The observation of the analyte is proportional to the number of moles or Stam 7 analyte present in the sample (na). Xa Na Xq = kna Sensitivity is the change in observation p' equivalent to the proportionality constant observation that can be measured, then the smallest difference in the amount of analyte that where k is the proportionality Const, er unit change in the amount of analyte ite it (k). If AX, is the smallest increment in i. can be given as Ana, Ana = a (total analysis method) ACa = ae (concentration method) Example: In particular analysis method, small increment in observation is + 0.0001 g ang if the method's sensitivity is 0.20 then _ £20002 _ 5 9.0905 9 4.2.4 Selectivity Selectivity is a measure of the method's freedom from interferences as defined by the method's selectivity coefficient. An analytical method is selective if its observation is a function of only the amount of analyte present in the sample. The selectivity of the method for the interferent relative to the analyte is given in selectivity coefficient Kay. Kai = k/ka where k; and kg are the sensitivities of analyte and interferent respectively, The selectivity coefficient may be positive or negative, it depends on the interferent. If the selectivity coefficient is > +1 or < - 1 indicates that, the method is more selective for the interferent than the analyte. 4.2.5 Robustness and Ruggedness In analysis, a used method must provide reliable results. This is our expectation; unfortunately, methods and their results are involved in the contribution of the uncertainty of various chemical and physical form interferences. When the method is relatively free from interferences, such methods are considered robust. The uncertainty in the result may be by random variation in experimental conditions. The tear of the method depends on experimental conditions like temperature, reaction PH, etc, and then it affects the result. If a method that is insensitive to changes in experimental conditions are called rugged. 4.2.6 Scale of Operation ere fg sees is an important term used in analytical chemistry. It gives a proper Sinead an imit for the selection of method, the proportion of concentration or See !e method, concentration, technique, and external conditions are "ot within the limit or in the scale of operation then we ace Its or do not 9% results. The limitations like proper amount of sample proper ‘concentration, and POPEalytical Chemistry -I (Sem. V) 46 _ Parameters of Instrumental Analysis f analyte should be mentioned. The scale of operation is shown in Fig. 4.3. Fig. 4.3 aft ee concentration of the analyte in the weight % on the y-axis and the sample size on shows wre ¥5- | ult trace 107% A-1g sample, 1% analyte a B- 0.1g sample, 10% analyte 10x C-0.01g sample, 100% analyte 10°% Trace a 10% 2 = 4 2 10°% g 10°% =| on | 1% | | 10% 100 % 1 04 0.01 g ee Mamoeni0y ol pol mg Meso 100 10 1 01 001 Kg eae 10 ng Microjgyc tt, Ultramicro <— ~log (weight of sample) Fig. 4.3: Scale of operation for analytical method The analytes are divided into major (>1% w/w), minor (0.01 - 1% w/w), trace ~ 0.01 % w/w) and below that ultra trace (<10-7 % w/w ), while the sample is divided into macro (> 0.1 g), meso (10 -100 mg ), micro (0.1- 10 mg) and ultra micro (< 0.1 mg). hoon diagonal lines shown in the graph are of a combination of sample size and ntration of an analyte. These lines help to define the limitations for analysis. ane Analysis sample size is 0.1 g i.e, macro analysis it requires major analysis. z Equipment, Time and Cost iia the finalization ; pare Concerning thei tions Cost of all chemic: Ment then it May reqi lo” of experimental determination, the analytical method can be i need of equipment, the time required to complete an analysis als, solutions, solvents, etc. for per sample. If we select a proper luire operator training as per the requirement.[Link]. Analytical Chemistry - I (Sem. V) 47 Parameters of Instrumental gy, The time required for the completion of an experiment also depends on the avai, all facilities in one place. Time also consumes for the preparation of standard soluti standardization of equipment to give reliable results. If the sample contains interferer’ material, then time is required for removing or masking such interference material, "he The cost of the analysis depends on many factors. It includes the cost of reagent, specific apparatus, necessary equipments, hiring charges if required, etc. 4.2.8 Making the Final Choice The criteria for the analysis of a particular determination gets finalized in terms considering the minimization of all errors. The errors get minimized to get reliable esults The result shows good accuracy and is most precise. The selected method should be able to detect the analyte in detection limit ie. smallest amount can be determined. The method Was also selected by considering the interferences present in the sample. The method and analytical procedures get finalized by considering the scale of operation level, time and cog required. 4.3 Developing the Procedure : Sees : Once we select the method, it is necessary to develop a procedure. In developing the Procedure, we have to consider the terms like calibration, standardization, sampling, validation and protocols. 4.3.1 Calibration and Standardization Calibration of equipment needs to be carried out on the regular basis. It is the process of determining its accuracy; it involves measuring observation from the equipment and comparing with standards. All measuring instruments, apparatus, and volumetric glasswares used in the experiment need to be calibrated to ensure they are offering accurate results. e.g. Balances are calibrated using standard weights whose mass can be traced to the internationally accepted platinum-iridium prototype kilogram. Standardization is the process of establishing the relationship between the amount of analyte and the method's observation or signal. For standardization of method, we use calibration graph for standard concentration of analyte with known concentration. The graph shown in Fig. 4.4 is known as calibration curve for standard. The method's observation changes with respect to the amount of analyte. abit io of Solvang Calibration graph (curve) Signal —» Cc, — Concentration Fig. 4.4: Calibration curve for standardization| Chemistry -I (Sem. V) ae Parameters of instrumental Analysis peewee 432 sampling : : E cample is @ portion of the material selected from a larger quantity of material. The of collection of samples is called sampling. a collected sample material, containing a small quantity represents a whole material | 1 litre water from the lake. This sample is called as gross sample. The gross sample further reduced to a small quantity is called a laboratory sample. From the laboratory sample, elect a very small quantity and taken it for the analysis is called an analytical sample or ener which is homogeneous and it must closely resembles the composition of the total nae of the material. aye Different sampling processes are used for solid, liquid, and gas samples. After collecting the analytical sample, the sample treatment is given like drying and dissolution in a suitable golvent as a stock solution. 4.3.3 Validation Validation is the process of verifying that a procedure yields acceptable results. It must be capable of producing results with acceptable accuracy and precision. There are many ways to validate analytical methods, such as, analysis by different analytical methods, analysis of the chemical composition of the test sample, analysis of individual analysis etc. Data validation is the final step before the release of the result. This process starts with validating the samples and methods used. 4.4 Protocol: Method documentation Standardization _— Control Establishment | chart | | of QC | Calibration * [Yes Validation A protocol j Fig. 4.5: Schematic diagram of protocol for analysis Used to acce . stringent written guidelines and specifying an exact procedure must be assurance Pt the results. When designing the procedure, a protocol contains quality and quality control procedures. The protocol contains instructions regarding the be Meth selection Internal calibration, Verificai Internal calibration blank[Link]. Analytical Chemistry - I (Sem. V) 49 Parameters of Instrumental Analysis quality assurance (QA) and quality control (QO) procedures. The steps are taken to ensure that the analytical work in given laboratory to good accuracy and precision considered under internal quality, while external quality for certification.vewemversag ws mewasgew wvamuut uuLerierence of another component. X Linearity : ¢ The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly Proportional to the concentration (amount) of analyte in the sample. A linear relationship should be evaluated across the range of the analytical procedure. For establishing linearity, number of working standards (dilute solutions) of increasing concentrations, prepared from standard stock solutions can be used. Linearity should be evaluated by visual inspection of a plot of signals (absorbance of workings standards) as a function of concentration of working standards. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares, correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. TECHNICAL PUBLICATIONS® - an up-thrust for knowledgea minimum of 5 concentrations (working standard solutions) is recommended. From a standard stock solution of a drug, diluted should be prepared in the range from 0 - 150 % of a expected concentration of a test sample ie. 30, 60, 90, 120 and 150 % of a test sample. Minimum three solutions of each concentrations are prepared and tested with a instrument. SK Range : e The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. For the establishment of linearity, The specified range is normally derived from linearity studies. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte. The following minimum specified ranges should be considered : o For the assay of a drug substance or a finished (drug) product: normally from 80 to 120 % of the test concentration; © For content uniformity, range covering a minimum of 70 to 130 % of the test concentration is recommended. © For dissolution testing : +/— 20 % over the specified range is recommended. © For the determination of an impurity: from the reporting level of an impurity 1 to 120 % of the specification;ere itt Limit of detection : The limit of detection of an indi ividual analytical procedure is the lowest amount i an be detected but not necessarily quantitated as an exact . analyte in a sample which c value. It is established by any one of the following approach - 1. Based on visual evaluation : Visual evaluation may be used for non-instrumental and instrumental methods. The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. 4 Based on signal-to-noise : This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low Concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. 3. Based on the Standard Deviation of the Response and the Slope : The detection limit (DL) may be expressed as- Detection limit = ae re ea ee(5-11) ec : The , —_Gallbration and Vari standard deviation of the resp Se conse ne Techniques where 5 $ = The slope of the calibration curve |. The slope S may be estimated from the calibrati ion curve of The estimate of o may be carried out ina variety of meee ways as : ;, Based on the standard deviation of the blank : + Measurement of the magnitude of yt A analytical background response is performed by analyzing an blank samples and calculating the standard deviation of a me number of sponses il, Based on the calibration curve : A specific calibration curve should be stud using samples containing an analyte in the range of DL. The enema deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. petimit of quantitation : + The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation. limit is a parameter of quantitative asa’ and is used particularly for the dete one of the following approach- may be used for both, instrumental ietermined by the ys for low levels of compounds mination of impurities and/or in sample matrices, degradation products. It is established by any i. Based on visual evaluation : Visual evaluation and non-instrumental methods. The quantitation limit is generally 4 ‘with known concentrations of analyte and by establishing the analysis of samples aini Jevel at which the analyte can be quantified with acceptable accuracy and Sheed yi iy on_ signal-to-noise {This approach can only be applied to analytical bit baseline noise. Determination of the signal-to-noise rao 'S n sured signals from samples with known low ¢ of blank samples and by establishing the alyte can be reliably quantified. A typical fe and the slope : The Quantitation isipstrumentation Techniques (5-11) ee o = The standard deviation of the Tesponse S = The slope of the Calibration cy, ve 0 , Theslope S may be estimated from the cays calibration curve Of the analyte, , The estimate of 6 may be carrie aa a j. Based on the standard deviatio f Inlety of ways as : s n of th analytical background response is perf blank : Measurement of the magnitud o itude of nk samp ; med by analyzi bla iples and calculating the Standard deviati Zing an appropriate number of ii. Based on the calibration curye f 10n of these responses. A i + A specific calibrati using samples i libration ci : siidika ch OMIM nate Be mgs of DL ‘Th ead ota i a regression li le resi : gression line or the standard deviation of dual standard lines may be used as the standard deviation, wee it of quantitation : . a quantitation limit 7 an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. ¢ The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. It is established by any one of the following approach i. Based on visual evaluation : Visual evaluation may be used for both, instrumental and non-instrumental methods. The quantitation limit is generally determined by the centrations of analyte and by establishing the analysis of samples with known cone: be quantified with acceptable accuracy and minimum level at which the analyte can precision. This approach can only be applied to analytical Determination of the signal-to-noise ratio is is from samples with known low 1d. by establishing the quantified. A typical Based on signal-to-noise : procedures that exhibit baseline noise. performed by comparing measured signal entrations of analyte with those of blank samples an conc minimum concentration at which the analyte can be reliably cise ratio is 10: 1. deviation of the resp‘ 10-1 : signal fitation the standard onse and the slope : The Quant iii, Based 0” Limit (QL) may be expressed as = a ——— ~~, TIONS® - an up-thrust for knowledgeCalibration and Validation Advanced Instrumentation Techniques (5- 12) ee 100 Quantitation limit = ae Where o = The standard deviation of the response S_ = The slope of the calibration curve ¢ The slope S may be estimated from the calibration curve of the analyte. The estimate of © may be carried out in a variety of ways, for example : i, Based on standard deviation of the blank : Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses. ii. Based on the calibration curve : A specific calibration curve should be studied using samples containing an analyte in the range of QL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. i. Robustness : @ The rahuctnece af an analytical nracedurea ie a manonen